Project description:We compare the brain and fin RNA-seq profiles of wild-type and scn8ab mutant zebrafish, which exhibit locomotion and fin regeneration defects.
Project description:Through molecular dynamics (MD) and free energy simulations in electric fields, we examine the factors influencing conductance of bacterial voltage-gated sodium channel NavMs. The channel utilizes four glutamic acid residues in the selectivity filter (SF). Previously, we have shown, through constant pH and free energy calculations of pKa values, that fully deprotonated, singly protonated, and doubly protonated states are all feasible at physiological pH, depending on how many ions are bound in the SF. With 173 MD simulations of 450 or 500 ns and additional free energy simulations, we determine that the conductance is highest for the deprotonated state and decreases with each additional proton bound. We also determine that the pKa value of the four glutamic residues for the transition between deprotonated and singly protonated states is close to the physiological pH and that there is a small voltage dependence. The pKa value and conductance trends are in agreement with experimental work on bacterial Nav channels, which show a decrease in maximal conductance with lowering of pH, with pKa in the physiological range. We examine binding sites for Na+ in the SF, compare with previous work, and note a dependence on starting structures. We find that narrowing of the gate backbone to values lower than the crystal structure's backbone radius reduces the conductance, whereas increasing the gate radius further does not affect the conductance. Simulations with some amount of negatively charged lipids as opposed to purely neutral lipids increases the conductance, as do simulations at higher voltages.
Project description:Therapies for cardiac arrhythmias could greatly benefit from approaches to enhance electrical excitability and action potential conduction in the heart by stably overexpressing mammalian voltage-gated sodium channels. However, the large size of these channels precludes their incorporation into therapeutic viral vectors. Here, we report a platform utilizing small-size, codon-optimized engineered prokaryotic sodium channels (BacNav) driven by muscle-specific promoters that significantly enhance excitability and conduction in rat and human cardiomyocytes in vitro and adult cardiac tissues from multiple species in silico. We also show that the expression of BacNav significantly reduces occurrence of conduction block and reentrant arrhythmias in fibrotic cardiac cultures. Moreover, functional BacNav channels are stably expressed in healthy mouse hearts six weeks following intravenous injection of self-complementary adeno-associated virus (scAAV) without causing any adverse effects on cardiac electrophysiology. The large diversity of prokaryotic sodium channels and experimental-computational platform reported in this study should facilitate the development and evaluation of BacNav-based gene therapies for cardiac conduction disorders.
Project description:Voltage-gated sodium channel Nav1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Nav1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Nav1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Nav1.6 in the presence of auxiliary subunits β1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the β1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Nav1.6-specific blockers. Structural mapping of disease-related Nav1.6 mutations provides insights into their pathogenic mechanism.
Project description:Voltage-gated Na(+) channels play an essential role in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. The recent solution of X-ray structures for the bacterial channel NavAb has provided an opportunity to study functional mechanisms at the atomic level. This channel's selectivity filter exhibits an EEEE ring sequence, characteristic of mammalian Ca(2+), not Na(+), channels. This raises the fundamentally important question: just what makes a Na(+) channel conduct Na(+) ions? Here we explore ion permeation on multimicrosecond timescales using the purpose-built Anton supercomputer. We isolate the likely protonation states of the EEEE ring and observe a striking flexibility of the filter that demonstrates the necessity for extended simulations to study conduction in this channel. We construct free energy maps to reveal complex multi-ion conduction via knock-on and "pass-by" mechanisms, involving concerted ion and glutamate side chain movements. Simulations in mixed ionic solutions reveal relative energetics for Na(+), K(+), and Ca(2+) within the pore that are consistent with the modest selectivity seen experimentally. We have observed conformational changes in the pore domain leading to asymmetrical collapses of the activation gate, similar to proposed inactivated structures of NavAb, with helix bending involving conserved residues that are critical for slow inactivation. These structural changes are shown to regulate access to fenestrations suggested to be pathways for lipophilic drugs and provide deeper insight into the molecular mechanisms connecting drug activity and slow inactivation.
Project description:The recent publication of a number of high resolution bacterial voltage-gated sodium channel structures has opened the door for the mechanisms employed by these channels to distinguish between ions to be elucidated. The way these channels select between Na(+) and K(+) has been investigated in computational studies, but the selectivity for Na(+) over Ca(2+) has not yet been studied in this way. Here we use molecular dynamics simulations to calculate the energetics of Na(+) and Ca(2+) transport through the channel. Single ion profiles show that Ca(2+) experiences a large barrier midway through the selectivity filter that is not seen by Na(+). This barrier is caused by the need for Ca(2+) to partly dehydrate to pass through this region and the lack of compensating interactions with the protein. Multi-ion profiles show that ions can pass each other in the channel, which is why the presence of Ca(2+) does not block Na(+) conduction despite binding more strongly in the pore.
Project description:Scn2a encodes voltage-gated sodium channel NaV1.2, a main mediator of neuronal action potential firing. The current paradigm suggests that NaV1.2 gain-of-function variants enhance neuronal excitability resulting in epilepsy, whereas NaV1.2 deficiency impairs excitability contributing to autism. This paradigm, however, does not explain why 20~30% of patients with NaV1.2 deficiency still develop seizures. Here we report a counterintuitive finding that severe NaV1.2 deficiency results in increased neuronal excitability. Using a unique NaV1.2-deficient mouse model, we found enhanced intrinsic excitabilities of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in Scn2a-related seizures. This increased excitability is autonomous, and is reversible by the genetic restoration of Scn2a expression in adult mice. RNA-sequencing revealed that the downregulation of multiple potassium channels including KV1.1, and KV channel openers alleviated hyperexcitability of NaV1.2-deficient neurons. This unexpected neuronal hyperexcitability may serve as a cellular basis underlying NaV1.2 deficiency-related seizures.
Project description:The voltage-gated sodium channel (VGSC) interacting peptide is of special interest for both basic research and pharmaceutical purposes. In this study, we established a yeast-two-hybrid based strategy to detect the interaction(s) between neurotoxic peptide and the extracellular region of VGSC. Using a previously reported neurotoxin JZTX-III as a model molecule, we demonstrated that the interactions between JZTX-III and the extracellular regions of its target hNav1.5 are detectable and the detected interactions are directly related to its activity. We further applied this strategy to the screening of VGSC interacting peptides. Using the extracellular region of hNav1.5 as the bait, we identified a novel sodium channel inhibitor SSCM-1 from a random peptide library. This peptide selectively inhibits hNav1.5 currents in the whole-cell patch clamp assays. This strategy might be used for the large scale screening for target-specific interacting peptides of VGSCs or other ion channels.
Project description:µ-Conotoxins are small, potent pore-blocker inhibitors of voltage-gated sodium (NaV) channels, which have been identified as pharmacological probes and putative leads for analgesic development. A limiting factor in their therapeutic development has been their promiscuity for different NaV channel subtypes, which can lead to undesirable side-effects. This review will focus on four areas of µ-conotoxin research: (1) mapping the interactions of µ-conotoxins with different NaV channel subtypes, (2) µ-conotoxin structure-activity relationship studies, (3) observed species selectivity of µ-conotoxins and (4) the effects of µ-conotoxin disulfide connectivity on activity. Our aim is to provide a clear overview of the current status of µ-conotoxin research.
Project description:Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na(+) ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.